This proposal is a response to PAR-12-058, Solicitation of Assays for High Throughput Screening (HTS) to Discover Chemical Probes (R01). This funding opportunity supports collaboration between academic, nonprofit, or commercial HTS screening facilities that have the requisite expertise and experience to implement an HTS-ready assay for the discovery and development of small molecule chemical probes. The work proposed will be conducted by collaboration between the laboratory that developed the concept of pharmacoperones (and the assay) and two other laboratories with extensive experience in HTS and access to large chemical libraries. We will use an existing and well-validated high throughput assay to identify pharmacoperones of the vasopressin type 2 (V2) receptor (V2R). The assay will be applied to screen a library of approximately 640,000 compounds and will identify therapeutic molecules for the treatment of misrouting of the V2R in nephrogenic diabetes insipidus, a rare and debilitating disease for which there are presently no effective drug treatments. Pharmacoperones are target-specific and small molecules that diffuse into cells, rescue misfolded protein mutants and restore them to function. Rescue is based on a newly appreciated mechanism: correcting the cellular routing of mutants that would otherwise be retained in the endoplasmic reticulum and unable to function. Because of the newness of this type of screen, this project will also serve as a prototype for the identification of pharmacoperones present in large chemical libraries. Accordingly, the proposed approach identifies drugs with a significant degree of novelty in therapeutic approach, relying on cellular mechanisms that are not currently represented in the Molecular Libraries assay pipeline; this technique offers an untapped opportunity for use of the HTS approach. Development of such assays is important and novel since useful chemical structures with the ability to control cellular trafficking may already be present in existing libraries, but have not been identified using existing screens. Preliminary data show that the assay was successfully transferred to the HTS facility and early screening results show that the proposed approach is likely to be successful in identification of hits.